Am J Respir Crit Care Med Vol 157. pp 76–80, 1998
Diagnosis of Nosocomial Pneumonia in Mechanically
Ventilated Patients
Repeatability of the Bronchoalveolar Lavage
PATRICK GERBEAUX, VERONIQUE LEDORAY, ALAIN BOUSSUGES, FLORENCE MOLENAT,
PHILIPPE JEAN, and JEAN-MARIE SAINTY
Service de Réanimation Médicale, Hôpital Salvator, Département d’Information Médicale, Hôpital Conception, CHRU Marseille, France
The repeatability of the bronchoalveolar lavage (BAL) was assessed prospectively in 44 mechanically ventilated patients with suspected nosocomial pneumonia. Two BAL were performed in the same lung area (contiguous segment) during two fibroscopic procedures performed with a thirty minute interval. All the bronchoscopies were performed by the same operator. The statistical analysis looked out for bias (MacNemar test), agreement, and repeatability (kappa test). In the 44 patients studied, the qualitative repeatability (i.e., presence or absence of bacteria) was excellent (95.4%). However, in the 16 patients having at least one positive culture, these results were more controversial. The quantitative repeatability for bacteria (same log10 for both BAL of the same patient) was the lowest of all the results (26.7%). The distinction between presence and absence of bacterial pneumonia (based on the 104 cfu/ml threshold) showed a repeatability of 75% with no bias, an agreement of 47% and a just-significant kappa test (test 5 1.97; p 5 1.96 for a 5% risk error). BAL seems to have excellent repeatability when sterile. Its repeatability when positive needs further studies to be
as-sessed. Gerbeaux P, Ledoray V, Boussuges A, Molenat F, Jean P, Sainty J-M. Diagnosis of
noso-comial pneumonia in mechanically ventilated patients: repeatability of the bronchoalveolar
lavage. AM J RESPIR CRIT CARE MED 1998;157:76–80.
Clinical and bacteriologic diagnosis of nosocomial pneumonia (NP) in mechanically ventilated (MV) patients is still difficult (1, 2). Different clinical criteria have been tested and have been proven to be inaccurate in predicting pneumonia (3, 4). Simple diagnostic procedures (such as endotracheal aspira-tion) give good qualitative information on tracheal ecology, but are unusable because of the frequency of the colonization of the proximal airways (5–10). Therefore, the past years have seen the development of new diagnostic methods such as pro-tected specimen brush (PSB) and bronchoalveolar lavage (BAL), in order to approach the reality of the pulmonary in-fection. Provided the use of threshold (103 cfu/ml for the PSB and 104 cfu/ml for the BAL), these new procedures are now recommended as reference methods in the diagnosis of NP in MV patients (6, 7, 11–14). However, autopsic studies report the persistence of false positive and negative results (15–19). According to scientific evaluation criteria, rigorous assess-ment of diagnostic methods is needed for efficient use (20). In this hierarchic procedure, the first of the five steps is the tech-nologic capability (i.e., the ability of the test to perform to specification in a laboratory setting). BAL and PSB have both
been tested under laboratory settings with good results. Moover, the clinical evaluation has raised the problem of the re-peatability of these procedures. A study on the reliability of the PSB showed that cautious intepretation of the results is advisable in clinical practice, especially if a decision (to treat or not to treat) is to be made (21). These results show how deep the gap can be between laboratory studies and clinical application. The clinical repeatability of the BAL has not been tested yet (20). Therefore, we decided to assess the repeatabil-ity of BAL in the diagnosis of NP in MV patients.
METHODS
Patients
Forty-four patients (32 men, 12 women) undergoing mechanical venti-lation (Servo 900 C, Siemens, Sweden) for more than two days and meeting criteria for suspicious pneumonia (i.e., new or progressive or persistent pulmonary infiltrate, purulent tracheal secretions, fever . 388 C) were studied. Mean age was 59 6 18 (SEM) years (range, 19 to 89). Mean SAPS was 11 6 4 (SEM) (range 4 to 24). The average time between starting of mechanical ventilation and the study was 12 6 11 (SEM) days (range, 3 to 57). The primary indication for ventilatory support included (Table 1): acute respiratory failure (n 5 20), exacer-bation of chronic obstructive pulmonary disease (n 5 11), acute car-diac failure (n 5 6), postoperative respiratory failure (n 5 4) and mis-cellaneous (n 5 3). For all the patients, the diameter of the tracheal prosthesis was superior to 8. Antibiotics were administrated to all pa-tients except for five of them, and for more than 3 days at the time of the study. All patients had a negative HIV status. This prospective study was approved by the Institutional Review Board (Comité Consultatif de Protection des Personnes dans le Recherche Biomedicale). Informed consent was obtained from the patients or the nearest relatives. (Received in original form April 19, 1996 and in revised form June 9, 1997)
Supported by the Département de al Stratégie et de la Recherche, Assistance Publique-Hopitaux de Marseille.
Correspondence and requests for reprints should be addressed to Patrick Ger-beaux, Service de Reanimation Medicale, Hopital Salvator, 249 Bd Sainte-Mar-guerite, 13009, Marseille, France.
Gerbeaux, Ledoray, Boussuges, et al.: Diagnosis of Nosocomial Pneumonia in Mechanically Ventilated Patients 77
Bronchoalveolar Lavage
Patients received intravenous premedication with midazolam and ve-curonium. Thirty minutes before the beginning of the first BAL (BAL1), the FIO2 was adjusted to 100% and was kept to this level until thirty minutes after the end of the second BAL (BAL2). During the same period, the positive end-expiratory pressure (PEEP) applied by the ventilator was equal to zero. No topical anesthesics were used. The trachea was suctioned before introducing the bronchoscope. The fiberoptic bronchoscope (FB 18X; Pentax, Tokyo, Japan) was intro-duced through a special adaptator (double rotule; Europe Médicale, Bourg, France) and advanced under direct vision next to the lobe set-ting the radiographic signs, without suctioning to avoid contamina-tion. The bronchoscope was then positioned in a segmental bronchus of the lobe. Three aliquots of 40 ml sterile normal saline were sepa-rately infused through the working channel of the bronchoscope. Hand suction was applied and the first aliquot withdrawn as bronchial lavage. The two other aliquots were pooled for bacteriologic and cyto-logic analysis. The samples were transported to the laboratory within 30 min of collection. The second bronchoscopy was performed under the same conditions 30 min after the end of the first one. The 30-min delay was necessary to sterilize the bronchoscope. Location of the sec-ond bronchoscopy was in the same lobe, adjacent segment. All the sam-pling procedures were performed by the same bronchoscopist in order to avoid operator-dependent variability.
Cytology
The differential count of macrophages, neutrophils, eosinophils, mas-tocytes, and contaminant elements (bronchial and upper aerodiges-tive cells) was made on centrifugation preparations of the BAL sam-ple. Two spots were prepared, in a Shandon spin, at 2,000 rpm for 2 min, one was stained with May-Grunwald-Giemsa, the other with Papanicolaou techniques; 200 cells were numbered on each spot. BAL samples were considered invalid when more than 1% of the cells counted were ciliated bronchial cells. For numeration counts, results are expressed as cells/ml of BAL.
Bacteriology
One aliquot of each BAL fluid was plated on MacConkey agar, choc-olate agar, and 5% sheep blood Columbia colistin-nalidixic acid agar (BioMérieux, Marcy l’Etoile, France). After 48-h incubation, colonies were counted and identified.
Statistical Analysis
The study tests the degree of agreement between two BAL (BAL1 and BAL2) performed on the same subject. Absence of systematic difference (i.e., bias) between the two BAL for the same patient was looked for using the MacNemar test (p . 0.05 meant no bias). The sults were then compared using the kappa test for agreement and re-peatability; p , 0.05 meant the test was repeatable (22–24).
RESULTS
The 88 BAL performed had less than 1% ciliated cells, assess-ing their alveolar origin. Usassess-ing the Wilcoxon test, no statistical difference was found between BAL1 and BAL2 for the total numerations (1,826,000 6 2,416,000 versus 1,036,000 6 1,737,000, respectively; z 521.55, p . 0.1) or neutrophilic PMN counts (1,491,934 6 2,123,469 versus 854,632 6
1,677,096, respectively; z 521.49, p . 0.1). Considering the qualitative repeatability of the cultures (i.e., the presence or absence of bacterial colonies), 42 patients (95.4%) had the same results on both BAL (Table 2). Twenty-eight (65.1%) were both negatives and 14 (31.8%) were both positives and showed the same bacteria. One patient had BAL1 positive and BAL2 negative, and one had BAL1 negative and BAL2 positive. The agreement level was 90% with no bias (p . 0.3 for MacNemar test, p , 1028 for kappa test). Applying the 104 cfu/ml threshold to the 44 pairs of BAL, 40 (90.9%) gave the same results, and the kappa test showed good agreement (72%, p , 1025) (Table 3).
The same statistical analysis performed upon the 16 BAL being at least once positive shows less significant results: 11 (78.6%) did not have the same log10 cfu/ml. The quantitative results were spread out on both sides of the 104 cfu/ml thresh-old in 4 (25%) of the 16 patients (Table 4). For these four pa-tients, numeration counts were controlled using the Wilcoxon test to assess absence of bias (i.e., one BAL was in pneumonia and not the other). Results showed no statistical difference be-tween positive and negative BAL for total numeration and neutrophilic PMN counts (Table 5). Upon the 16 patients, the agreement level was 45.5% with no bias (p . 0.3 for MacNe-mar test). The kappa test showed limited significance (test 5 1.97; p 5 0.05 for test 5 1.96). The intrasubject variability has been studied for agreement (Figure 1). The same log10 cfu/ml was found in five patients (agreement: 31.3%, p . 0.15 for kappa test). The difference in log10 cfu/ml was one (or less) in 11 patients (agreement: 75%, p , 1029), two (or less) in 13 (agreement: 87.5%, p , 1029) and three (or less) in 16 (agree-ment: 100%, p , 1029). The order of passage of the BAL did not influence the quantitative results (BAL1 . BAL2: 7, BAL1 5 BAL2: 5, BAL1 , BAL2: 4, MacNemar test: p . 0.85). No complication or respiratory distress was seen.
DISCUSSION
The BAL is one of the two methods recommended to assess the diagnosis of nosocomial pneumonia in ventilated patients TABLE 1
PRIMARY INDICATION FOR VENTILATORY SUPPORT
Acute respiratory failure 20
Exacerbation of chronic obstructive pulmonary disease 11
Acute cardiac failure 6
Postoperative respiratory failure 4
Miscellaneous 3
Total 44
TABLE 2
RESULTS OF THE CULTURES OF THE BAL
BAL1
Recovery of Bacteria Sterile Culture Total BAL2
Recovery of bacterias 14 1 15
Sterile culture 1 28 29
Total 15 29 44
MacNemar test 5 nonsignificant. Kappa test: p , 1028.
TABLE 3
RESULTS OF THE CULTURES OF THE BAL WITH THRESHOLD
BAL1
Infection No Infection Total BAL2
Infection 8 1 9
No infection 3 32 35
Total 11 33 44
Infection 5 presence of bacteria ù 104 cfu/ml.
No infection 5 no bacteria or presence of bacteria , 104 cfu/ml.
78 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 157 1998
(2, 18, 20). Its accuracy is dependent on the patients’ charac-teristics, the operator and sampling methods, and the inter-pretation of the results (25–27). Because of the bronchial con-tamination, a recommended diagnostic threshold has been established (104 cfu/ml) for the bacteriologic results (6, 7, 11– 14). One could debate this 104 cfu/ml threshold: 103 cfu/ml is more sensitive (but less specific), while 105 cfu/ml is more spe-cific (but less sensitive). The 104 cfu/ml threshold appears to be the best compromise between sensitivity and specificity. In any case, the use of these measurements in daily practice (for diagnostic purposes and therapeutic decisions) assumes that their repeatability is acceptable. Bacteriologic results of the BAL (using the 104 cfu/ml threshold) are important for the cli-nician, for therapic decisions (to treat or not to treat). And that decision is an important one. Treating someone who does not need it can lead to nosocomial infection with antibiotic-resistant bacteria, or to important side effects. Not treating someone who needs it can lead to death. So this result has to be reliable in daily practice. Many variations among the posi-tive results have been noted. The explanations for these varia-tions are not clear. All the procedures were performed by the same operator, in order to avoid operator-dependent factors. All the samples were studied in the same laboratory as usual, the only difference being that nobody but the fibroscopist knew which BAL was the first one or the second one. Intra-subject variability can be ruled out because the patients were their own control. Repercussion of the first BAL upon the
quality of the second can be discarded because of the absence of bias. Variations of dilution of the bronchial secretions could account for the variability of the qualitative or quantitative culture results. However, total cellular counts and neutro-philic PMN counts were not statistically different between BAL1 and 2. Moreover, BAL were performed as they are in daily practice, and these results seem to us applicable to clini-cal practice. As in daily practice, most of the patients were re-ceiving antibiotic therapy (88%). It is important to notice that those antibiotic therapies were the same for at least 3 days, so that it was licit to suspect a resistant-bacteria pneumonia, and to perform a BAL. The aim of this study is to test the repeat-ability of the BAL by repeating the sampling procedure twice in the same lobe (contiguous segments) in MV patients with suspected NP. The results were compared using the MacNe-mar test, which looks for a systematic difference between two results. In this study, the MacNemar test appreciates the pres-ence of a bias. The MacNemar test is not significant in our re-sults. This fact allows us to say that there is no bias reliable to the technicality in this study. As there is no bias, the kappa test may be used (22–24). It looks for repeatability. It calcu-lates the agreement level (in percentage), but as it is depen-dent on the prevalence of the factor studied, it is not a good statistical result. Using the test itself, it gives a statistically use-ful result. In this study, the repeatability is statistically demon-strated when p , 0.05 for kappa test. As shown in this study, absence of pneumonia (based on presence of bacteria in the TABLE 4
CULTURE RESULTS OF THE TWO BRONCHOALVEOLAR LAVAGE (BAL)
Patients Bacterial Species
BAL1 (log10 cfu/ml) BAL2 (log10 cfu/ml) Concordance with Threshold 104 cfu/ml
5 Pseudomonas aeruginosa 5 4 Concordant
9 P. aeruginosa 4 6 Concordant
11 Staphylococcus aureus 0 2 Concordant
13 P. aeruginosa 6 5 Concordant
14 Pseudomonas mirabilis 3 0 Concordant
16 P. aeruginosa 2 3 Concordant 18 P. aeruginosa 4 3 Discordant 23 P. aeruginosa 6 6 Concordant 24 P. aeruginosa 6 6 Concordant 31 S. aureus 6 5 Concordant 34 E. cloacae 3 4 Discordant 37 P. aeruginosa 4 3 Discordant 40 P. aeruginosa 2 2 Concordant
41 Escherichia coli 4 4 Concordant
43 P. aeruginosa 5 2 Discordant
44 P. aeruginosa 5 5 Concordant
From 1 to 44, patients not named had sterile cultures.
TABLE 5
NUMERATION COUNTS OF THE BAL OF THE FOUR DISCORDANT PATIENTS
Patient Number Cell Counts in BAL (infection) Cell Counts in BAL (no infection) Neutrophil Count in BAL (infection) Neutrophil Count in BAL (no infection) 18 600 1,000 552 540 34 800 1,500 672 768 37 5,000 170 4,850 4,800 43 2,600 7,500 2,418 2,392 Mean (6 SD) 2,250 (6 2,042) 2,542 (6 3,350) 2,123 (6 2,008) 2,125 (6 1,964) Wilcoxon test Z 520.73 p . 0.45 (NS) Z 5 20.365 p . 0.71 (NS)
SD 5 standard deviation. NS 5 nonsignificant. Counts expressed as 103
Gerbeaux, Ledoray, Boussuges, et al.: Diagnosis of Nosocomial Pneumonia in Mechanically Ventilated Patients 79
BAL with less than 104 cfu/ml) is a highly reproducible result (p , 1025). But presence of pneumonia is a poorly reliable re-sult (0.04 , p , 0.05). If one considers that, given the impor-tance of the result of BAL, on therapic decision, the threshold for significance has to be 1% (p , 0.01), it appears that BAL is not reproducible when positive. The intrasubject variability shows that only 31.25% of the positive BAL have the same log10 cfu/ml. A variability of one log10 cfu/ml has to be allowed for the BAL to be reliable. That low degree of repeatability could account for the different results that different authors express in the literature. However, this result is limited be-cause of the small number of positive patients. BAL seems to be a reproducible diagnostic method, especially when bacte-rial cultures are negative. The limited significant threshold when cultures are positive leads us to recommend cautious in-terpretation in daily practice. Further studies are needed be-fore definite conclusions can be drawn.
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